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Most animals usable for collection are not close enough to our facility and therefore, it becomes necessary to develop ways to collect and bring semen samples to our laboratory while maintaining pre-freeze and post-thaw quality. Under ideal circumstances a 48-hour time span from collection to freezing would satisfy our needs and those of commercial stud service operations because the same barriers to efficient collection, processing, and freezing ram semen for artificial insemination are encountered by all.

Semen from bulls, boars, and stallions can be cooled and maintained at low temperatures, depending on species, before freezing and still result in acceptable post-thaw sperm quality and fertility. Ram sperm held at low temperatures for multiple days-but not frozen-maintains acceptable motility (exhibits spontaneous movement) and fertility, but the effects of holding ram semen up to 48 hours-and then freezing-have not been reported. So this study’s goals were to

Determine the feasibility of holding ram semen prior to freezing and

Determine what effects this would have on the post-thaw quality and fertilizing potential.

How The Experiment Was Carried Out

Semen Collection, Pre-Freeze

Evaluation & Cryopreservation

Semen was collected from six rams in the autumn and from six different rams in the spring using electroejaculation. Sperm concentration (number of sperm per ml of semen) and motility were estimated to ensure sufficient numbers of sperm and reasonable quality, respectively.

All samples had an initial total motility of at least 70%. The samples were then diluted in one step with 37°C (98.6°F) egg yolk-Tris medium (Tris is a buffering agent that resists acid/alkaline changes.) containing antibiotics so that the final concentration was 400 million sperm per ml.

The samples were cooled to 5°C (41°F) over period of 2 hours, where they remained until freezing.

Equal volumes of the samples were loaded into artificial insemination straws at “Time 0″ (immediately upon reaching 5°C/41°F). More straws were loaded at 24 and 48 hours after “Time 0.” The straws were frozen in liquid nitrogen vapor and then plunged into liquid nitrogen for storage.

Post-Thaw Analysis

The post-thaw sperm membrane quality was also evaluated using flow cytometry (assessment of moving cells).

An illustration of mammalian sperm that identifies the various regions, organelles, and membranes of the cell.

For this type of evaluation sperm are stained with fluorescent probes, which-when excited using lasers in the flow cytometer-label different identifiable sperm characteristics and consequently provide information about the state of the sample as a whole at a rate of 50,000 sperm per minute.

In this instance fluorescent probes were used to evaluate

Integrity of plasma membrane (a membrane that covers the entire sperm surface) and

Integrity of acrosome membrane (a membrane on the head of the sperm, which contains a number of enzymes used for penetrating the egg; see Figure 1).

By analyzing sperm in this manner, four types of sperm populations can be detected:

Dead sperm that are non-acrosome reacted,

Dead sperm that are acrosome reacted,

Live sperm that are non-acrosome reacted, and

Live sperm that are acrosome reacted can be detected.

If there is a large percentage of sperm with damaged plasma membranes, or acrosome membranes, or a combination of the two, it is indicative of dead or poor quality sperm, respectively and therefore a poor sample.

Membrane Binding Analysis

The ability of frozen-thawed ram sperm to bind to the membranes of store-bought hen’s eggs was also evaluated as a test of the overall quality of a sperm sample.

The rationale for this experiment is that multiple aspects of the fertilization process can be approximated using this test. Other research has utilized this method for evaluating mouse, bull, horse, rooster, and human sperm (Barbato et al., 1998).

In this analysis chicken egg yolks are separated from the whites and the yolk sack membrane is opened to release the contents. The membrane is washed until it is free of yolk and then cut into uniform pieces. The frozen-thawed sperm is then mixed with each membrane and incubated at 39°C (102.2°F), the temperature of a female mammalian reproductive tract.

After 1 hour of incubation the samples were stained similar to the flow cytometry analyses and incubated for an additional hour.

The membrane samples were washed to remove unbound cells, placed on a microscope slide and the number of sperm bound to each membrane was determined using a Nikon fluorescent microscope.

This analysis was replicated three times for each cryopreservation time per ram, using membranes from three different eggs. The processes of capacitation (“ripening” of sperm, becoming able to fertilize eggs) and the acrosome reaction can be evaluated by analyzing the sperm samples in this manner. Only sperm that have undergone these processes are capable of binding to a membrane. As the same proteins used for binding mammalian sperm to mammalian eggs are present on hen’s eggs, binding is possible and accordingly, this becomes a very economical and simple method for analyzing sperm binding ability.

Results, Discussion, Conclusions

Holding sperm in this manner did not affect its post-thaw motility, progressive motility, plasma membrane integrity or acrosome membrane integrity in either of the seasons analyzed. Holding sperm in the manner described here at low temperatures before insemination, freezing, or both, is commonly done in many species with acceptable results.

Other researchers have demonstrated that ram sperm can be held prior to freezing, which results in either no change in post-thaw motility when held for six hours (Patt and Nath, 1969) or a modest decrease of about 5% after 18 hours of holding (Fiser and Batra, 1984). Because of these findings, it was hypothesized that holding ram sperm for 24 to 48 hours would have a negative effect and cause a decline in the overall quality of the sperm.

Therefore, it was surprising to observe that the holding time resulted in no significant declines in motility, plasma membrane integrity, or acrosomal membrane integrity of live sperm at the three holding times tested. These findings are cautiously stated because it is known that the quality of sperm declines over time due to the fact that sperm do not contain a means of self reparation. Furthermore, these analyses are very limited in the information they provide in that no one test completely evaluates the quality of a sperm sample.

The results from the membrane binding experiment are of interest because no differences were observed across holding times in autumn. In contrast, the mean number of sperm bound at the 48-hour holding time in spring had significantly more sperm bound than the “Time 0″ spring holding time but Time 24 was not different from either the “Time 0″ or 48.

The differences in membrane binding may possibly be due to a difference in ram seminal plasma composition and/or membrane structure due to the spring season and the fact that rams are traditionally seasonal breeders (Evans and Setchell, 1979). This may make the sperm more readily able to fuse with a membrane in an in vitro environment and consequently account for the differences in membrane binding.

One might want to utilize the membrane binding assay to determine fertility potential of a ram or semen sample because differences in rams were observed (data not reported) and because it has been utilized for this purpose (Gill et al., 1999). This assay is more properly used for evaluating the potential for a sperm sample to capacitate and acrosome react; processes essential for in vivo fertilization. Still, these results demonstrate that holding time does not prematurely induce capacitation and the acrosome reaction and consequently result in lower quality sperm.

Comparisons of season were not performed in this research because different groups of rams were used in the different seasons and analyses were performed at two different times of the year. Therefore direct comparison would not have been appropriate. These results are not surprising though, because this methodology has been used to detect differences in males (Gill et al., 1999) and male to male semen differences are known to exist.

Together, this research demonstrates that ram sperm can be collected and held for up to 48 hours prior to freezing without a negative impact on the post-thaw sperm quality. These techniques can therefore be used by producers to have semen samples frozen at commercial collection services that are not within a convenient distance of their farm or ranch. Currently, research is being conducted to determine the actual effects on fertility (using artificial insemination) of holding ram sperm for extended periods of time at 5°C (41°F) prior to freezing.

This methodology has proven useful to the NAGP and enabled us to collect semen samples from around the country.

While some of the material in the NAGP has been donated already frozen, a large number of the insemination doses have been frozen using these protocols.